Contributions to the Evolution of Blood Pressure Regulation

Z. klin. Chem. u. klin. Biochem. 7. Jg., S. 464—466, September 1969

Contributions to the Evolution of Blood Pressure Regulation Part II: Evidence for the Absence of Kinin-like Polypeptides Released by Proteolytic Enzymes for Blood Pressure Regulation in Fish

By ROSMARIE VOGEL, H. SCHIEVELBEIN, W. LORENZ and E. WERLE, with the assistance of A. SCHMAL From the Institut fur Klinische Chemie mid Klinische Biochemie der Universitat Miinchen (Direktor: Prof. Dr. Dr. E. Werle)

(Fingcgangen am 9. Mai 1969)

The intravenous injection of Padutin, bradykinin, kallidin and eledoisin is without effect on the blood pressure of cartilagenous and teleost fish. Preparations of fish pancreas and fish serum prepared in the same manner as organs of mammals and birds for prekallikrein/ kallikrcin or kininogen/kinin were also without effect. Therefore in fish the existence of a kallikrein-kinin like circulation regulating system can be excluded.

Die intravenose Injektion von Psfdutin, Bradykinin, Kallidin und Eledoisin hat keine Wirkung auf den Blutdruck bei Knorpel- und Knochenfischen (Katzenhai und Wels). Auch Praparate aus Fischpankrcas und Fischserum, die gleichermaftcn wie Saugctier- und Vogclor- ganc aufgearbcitct waren und demnach PraekalHkrein bzw. Kallikrcin oder Kininogen bzw. Kinin enthalten muftten, waren ebenfalls wTir kungslos. Die Existenz eines blutdruckwirksamen, Kallikrein-Kinin-ahnlichcn Systems kann demnach bei Fischcn ausgeschlossen werden.

In the first part of this paper we described the presence fresh water. Acetone — dried powder was prepared by mixing one of an adrenergic system in cartilagenous and teleost part of pancreas homogenate with one part of acetone; drying was performed at room temperature. fish (1). Preparation of a kinin-like component from fish: serum from In mammals and birds in addition to the adrenergic fish was incubated with fish pancreas homogenate or trypsin system for blood pressure regulation, there exist two according to known methods (5). different enzymic regulation mechanisms, which are in• The sensitivity of the test animals to vasoactive substances was dependent of each other. These systems are the renin- always tested with adrenaline and histamine respectively (fig. 1). angiotensin system and the kallikrein-kinin system. Results Phylogenetic investigations are described for the renin- angiotensin system especially with regard to the releasing Kallikrein of mammalian origin (2—20 units/kg body enzyme (2, 3). Investigations with regard to the kalli• weight) had no influence on the blood pressure of fish. krein-kinin system led to the detection of a kallikrein (One tenth of the applied minimal dose lowers the blood inhibitor in fish serum (4). The components of the pressure of the dog to approximately 40% of the normal kallikrein-kinin system are very species-specific (5, 6), value). with respect to the releasing enzyme (kininogenase) and Fish pancreas homogenate (corresponding to 2—20 mg also to the pharmacological effect of the kinin. tissue applicated/kg) induced an irreversible dose-inde• In this paper we describe investigations to find evidence pendent decrease in blood pressure of 2—3 mmHg (see for the presence or absence of the kallikrein-kinin system fig. 1). This slow response could not be eliminated by in fish, using methods already proved for the differen• boiling or incubation of the homogenate with Trasylol; tiation of the components of the system. it is caused by the sedimentable fraction of the homo• genate. On the other hand, the depressor effect cannot be demonstrated, following the application of known Methods and materials treatments for the enrichment or activation of kallikrein, The methods used for measuring the blood pressure in fish are e g. dialysis or acetone precipitation of the homogenates. the same as described in part I of this paper (1). As a representative Occasionally a pressor effect was observed (see fig. 2). of the cartilagenous fish wc used the catshark (Scyliorhinas steltaris) The application of trypsin led in the fishes to a slow and as a representative of the teleost fishes the catfish (Silurus decrease in blood pressure. The irreversible decrease to glanis). minimal pressure resembled the circulatory failure in Substances and drugs used: adrenaline (Suprarenin Hoechst), noradrenaline (Artercnol, noradrenaline hydrochloride Hoechst), mammals induced by shock.

histamine (Imido, Roche), acetylcholine (Roche), bradykinin Kallidin y bradykinin and eledoisin had no effect on the blood synth. (Sandoz), kallidin synth. (Sandoz), eledoisin (Sandoz), pressure of fish up to doses of 20 //g/kg. (In mammals trypsin (Worthington), polyvalent proteinase inhibitor (Trasylol, Bayer), renin (isorenin preparation according to (7)), Val-5- 1/100 part of this dose is effective.) angiotensin I (Ciba), Val-5-angiotcnsin II (Ciba), hog pancreas Fish serum prepared for the identification of kininogen- kallikrein (Padutin, Bayer). kinin mechanism (see methods) had no depressor effect in Preparation of a kallikrein-like component from fish: pancreas circulation of fish. On the contrary, a pressor effect was homogenates from shark, catfish and tench (finea vulgaris) were observed (see fig. 1). prepared by the homogenisation of one part tissue with 100 parts water. The extracts were used as such, or first separated into Renin in a dosage of 5 mU or 50 Goldblatt-U/kg (for supernatant and precipitate, or first dialyzed for 15 hours against the preparation, see methods) had no effect on the blood (1 min ^

3* 3>

Fig. 1 Absence of components of the kallikrein-kinin system in fish Catfish, 1,370 g; urethane anaesthesia; blood pressure in the caudal artery registered with Statham transducer (see methods in SCH i EVELBEI N and coworkers (1)) Injections in the caudal vein: 3.6 mg/kg pancreas of catfish homogenized 1:100 (raster: control volume) 5: 3.6 jug/kg noradrenaline 3.6 //g/kg acetylcholine 6: 0.7 m//kg tench serum incubated with homogenate from 3.6 /

100

Fig. 2 Absence of kallikrein in fish pancreas Catfish, 4,000 g; urethane anaesthesia; blood pressure in the caudal artery registered with Statham transducer (see methods in SCHIEVELBEIN and coworkers (1)) Injections in the caudal vein: 1; 5 //g/kg adrenaline 2: Acetone-dried powder from catfish pancreas corresponding to 125 mg fresh tissue/kg body weight fish pressure in the species used; angiotensin I (10—20 jugj gate the possible existence of a species-specific renin- kg) was also ineffective. Angiotensin II (7—20 ^g/kg) led angiotensin system; evidence was obtained, however, to a prolonged stimulation of respiration similar to the for the liberation of pressor substances refered to by effect of great doses of nicotine. WEICHERT (2) and MALVIN and VANDER (3). There can be several causes of the observed retardation of responses and the different dose-response relationship Discussion in fish as compared to mammals. One of these reasons All methods known to us (5) failed to demonstrate the may be the occurrence of analogues of derivatives of cate• existence of the kallikrein-kinin system or components of cholamines (SCHIEVELBEIN and coworkers (1)). Finally this system in cartilagenous and teleost fish. The only the special anatomical conditions of the fishes can be in• known components arc the fish serum inhibitor, which volved. According to ROMER (11), in fish the so-called was detected by WERLE and coworkers, 1950 (4) and a renal-hepato circulation with a widespread capillary kininase found by ERDOS and coworkers (8). But the system interrupts the caudal vein, which we and other inhibitor may be an unspecific proteinase inhibitor for investigators (12, 13) used for application of the sub• the regulation of proteolytic reactions known in species stances tested. Especially with regard to the components of lower ordines (9, 10) and the kininase may be an of the kallikrein-kinin system, these conditions may have unspecific carboxypeptidase. The observed depressor led to a loss of the substances by inactivating enzymes or effect of pancreas homogenate cannot be attributed to a tubular secretion and subsequent excretion (5). kininogenase (kallikrein) because after enrichment of With regard to this possibility we investigated the vascular high molecular components this effect was no longer conditions in the catfish, which reacted essentially in the observed. same manner as the shark. Figure 3a is a X-ray picture of Angiotensin I and II and a mammalian releasing enzyme an endoradiography of a part of the venous system im• had no effect on the blood pressure. We did not investi• mediately after injection of contrast medium, the volume of which corresponds to the usual applied volume. the existence of a kinin-libcrating system comparable to As can be seen, the contrast medium immediately ad• that of mammals and birds. In spite of this there may oc• vances to the heart. From figure 3b there can be seen the cur free kinin-like peptides because in venoms of species beginning of the distribution of the contrast medium of lower orders a number of preformed kinins were ob• into the capillary system of the kidneys. These findings served (for review see PISANO, 14). Perhaps there is no are evidence for the fact that the passage of substances comparable regulating system in lower animals. from the caudal vein to the common circulation is not These investigations were partly performed at the "Naples Zoolo• hindered and that the circulation of the kidney is ef• gical Station*', and \vc are very grateful to Prof. Dr. E. ROCCA fected by a shunt. From the fact that all substances and Dr. R. MARTIN for valuable advice. studied in part I and II of this paper were tested in both We acknowledge financial support by the Deutsche Forschungs- fish species with the same results, it can be concluded gemeinschaft. We are indepted to Prof. Dr. M. POSCIIL, Abt. f. Rontgenologic that the absent or slow reactions are real results. und Strahlenthcrapie der Chirurgischcn Universitatsklinik Miin- In cartilagenous and teleost fishes there are no hints for chen for the opportunity to perform the angiographics.

References

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